Oxford Nanopore announces technology updates at Nanopore Community Meeting
Wed 1st December 2021
Nanopore Community Meeting, Oxford, UK
Highlights include release of new “Kit 12” and R10.4 flow cells for >Q20 (>99%) raw read and around Q30 (99.9%) Duplex sequencing accuracy, and the new PromethION P2 device, a palm-sized, high-throughput sequencer that delivers the most accessible low-cost, high-output Oxford Nanopore sequencer
Oxford Nanopore has provided a technology update at its Nanopore Community Meeting, describing a range of product releases and upgrades. These include the release of “Kit 12”, a new kit that includes the latest “Q20+” chemistry and enables “Duplex” sequencing. This is coupled with the release of R10.4 flow cells. Users of nanopore sequencing can now achieve >Q20 raw read/“simplex” accuracy or around Q30 Duplex accuracy, and enhanced, high-accuracy consensus sequencing and variant calling, using these released products.
The Company has also announced PromethION 2 (P2), a device that can run up to two high-throughput PromethION Flow Cells, which will be available in starter packs from US$10,455. The P2 is designed to bring low-cost nanopore sequencing for large genomes or high-throughput long-read transcriptomics, without the requirement for capital investment and with minimal infrastructure requirement. P2 will be available as a standalone device with integrated compute, or “P2 Solo”, a sequencing device that can be connected to existing compute, including GridION. P2 Solo will be shipped from Q2 2022 and Oxford Nanopore is now taking pre orders here.
Oxford Nanopore also introduced Remora, a new tool for methylation analysis. Remora further enhances base-modification analysis, and is used during the experiment (concurrent to standard basecalling), at no additional cost, while maintaining high-accuracy basecalling of native bases. Remora provides industry-leading performance at only 20X coverage.
“Short fragment mode” (SFM), to optimise accurate, high-throughput sequencing of shorter fragments as short as 20 bp, will be fully enabled in the new year. Oxford Nanopore’s technology sequences DNA or RNA molecules of any length, from short to ultra-long. However, software output settings have historically avoided shorter “adapter only” reads. SFM is designed to optimise applications where larger volumes of shorter fragments require high-accuracy analysis, supporting as many as 250M native human reads, at ~200 bp, on a PromethION flow cell.
“Path to in-field liquid biopsy”. R&D pipeline programmes were reviewed, including chemistries such as “inny” and “outy”, that capture a single molecule of DNA and make multiple measurements of that single molecule. These techniques have the potential to deliver “Adaptive Accuracy” – supporting capture and re-reading of rare variants in a mixed sample. could be paired with liquid biopsy workflows in the lab or in the field, potentially enabling easy-to-use remote “liquid biopsy” sequencing.
The Company is developing an integrated sequencer and tablet; the MinION Mk1D, that would be capable of processing the high data volumes produced by MinION and could be used for this purpose.
Nanopore Community Meeting
The annual Nanopore Community Meeting, held online in 2021, has this year featured more than 50 user talks, spanning areas including human genetics, clinical / cancer research, microbiology including genomic epidemiology, plant and animal research and bioinformatics. Write ups and videos of the talks are made available on the nanopore website. At the meeting, Oxford Nanopore provided a technology update, featuring Clive G Brown (CTO) and talks from James Clarke, Stuart Reid and Rosemary Sinclair Dokos. In addition to the above, the following updates were presented:
Users can now choose between flow cells that use the R9.4.1 nanopore, paired with “Kit 10 or Kit 11”, which run at ~420 bases per second. They may also choose the new R10.4 nanopore, a newer design with longer reader head that is designed to provide high resolution of homopolymers and increased consensus accuracy. R10.4 is paired with the new Kit 12, which runs at ~230-270 bases per second. Kit 12 enables the newest “Q20+” chemistry and “Duplex”, where two complementary molecules from the same Duplex strand are sequenced consecutively for higher accuracy.
With an additional choice of basecalling modes, users can tailor their experiments to their requirements, and achieve greater than Q20/99% raw read accuracy on fully released products. Single molecule Duplex read accuracies are distributed around Q30/99.9%, with the longest Q30 read demonstrated so far at 156 kbases (although there is no fundamental limit here).
This raw read accuracy performance provides the foundation for high consensus accuracy, which can reach Q50 (99.999%), and the ability to detect and characterise a range of genetic variants including single point mutations, structural variations and methylation throughout the entirety of the genome, for the most comprehensive genomic analysis.
Oxford Nanopore provided an update on the latest analysis tools for optimised performance, including recommending Medaka for consensus accuracy of >Q50 (bacterial), Clair3 for SNP and Indel calling (F1 scores of 99.9% and 89% respectively, representative of whole genome performance) and LRA/CuteSV for structural variation (F1 96.1%).
Oxford Nanopore announced that base caller models now include native Plant DNA as part of their training, such that plant-specific base modifications present are recognised as canonical bases as opposed to errors, increasing the accuracy for users sequencing highly modified plants. Modifications can be recovered from the signal using Remora.
- A new upgrade of PromethION chip – the Marathon chip (M chip) – will be released in early 2022, providing greater output through extended run duration, and PromethION compute towers will also be upgraded for increased compute capacity
- Potential pipeline chemistries were reviewed, including “inny” and “outy” and the potential to deploy “Adaptive accuracy” – real time sequencing until a target accuracy has been achieved, supporting high-accuracy sequencing of rare variants in single molecules, in a highly efficient way.
- These pipeline chemistries have the potential to support ‘liquid biopsy’ workflows that could detect and analyse molecules from various samples such as blood, saliva or environmental samples, either in a lab environment or in the field. To accompany these chemistries, Oxford Nanopore is developing a mobile device designed to sequence direct from sample. No timeline has been provided for this device, which has the potential to also be adapted for other analytes such as small molecules.
- Progress was announced in the development of a low-cost flow cell for Flongle, using a polymer substrate instead of silicon. With proof of concept at 120 nanopores per chip, on which 2 Gb of data has been generated. This design is now being moved into a prototype manufacturing phase.
- The first sequencing data was presented from the new generation ASIC, a new, smaller, low power and low noise design of electronic chip designed to drive the development of new products such as the SmidgION sequencer for mobile phone or Plongle desktop “array sequencer”.
Multiple sample preparation updates were provided:
- The launch of Kit 12 and R10.4 for highest accuracy sequencing is complemented by a native barcoding kit so that the Q20+ chemistry can be multiplexed, and is also compatible with the rapid kit for Q20+
- The Monarch NEB ultra-long sequencing protocol was launched in the summer to enable ultra-long read sequencing, compatible with protocols on cell and blood and now tissue extraction.
- For high-throughput sequencing on PromethION, the new multiplex ligation kit is now in Early Access and is intended to deliver two human genomes per PromethION flow cell with the capacity to sequence up to 96 samples using the latest barcoding kits
- Automation for high throughput sequencing operations is being further developed, including Hamilton script for two genomes per PromethION flow cell
- VolTRAX devices that are PCR-enabled are now shipping to customers
- The Midnight kit, for sequencing SARS-COV-2 for COVID surveillance, in 5.5 hours and for as little as $9.55, was launched in the summer. It continues to detect Omicron and successfully identify lineage. Further updates are bring provided to users in response to the evolving COVID situation
- The rapid/field kits can now support inputs of 50-100 ng of DNA, down from 400 ng; this improvement is being developed for the whole range
- The new cDNA PCR kit is also launched, which can deliver more than 20M 1 kb reads on MinION flow cells and over 160M reads on PromethION flow cells. This kit also simplifies single-cell transcriptome analysis, with the ability to deliver full-length reads for isoform discovery/characterisation, gene fusions, cell-subtype analysis or spatial profiling
- Adaptive Sampling is a unique feature of Oxford Nanopore sequencing platforms, enabling real-time selection of DNA regions of interest. In addition to target enrichment, Oxford Nanopore also demonstrated reduced representation methylation sequencing using MinKNOW. The ability to obtain 6 million CpGs from a single MinION run, represents superior performance to bisulfite methods, with the usual nanopore benefits of simple workflow, low cost and time-to result.
- Updates were provided on developments in software, including the expansion of EPI2ME into regional areas, to support broader access to ‘plug-and-play’ analysis workflows
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